i. Field of the Invention
The present invention relates to a fluorometric method for assaying primary amines and more particularly, to a class of compounds which react with primary amines to form fluorescent adducts.
ii. Description of the Prior Art
Assaying techniques wherein a fluorogenic reagent is reacted with a substrate to form a readily detectable fluorescent moiety have been known for some time. One fluorogenic reagent which has been employed for assaying primary amines is o-phthaldehyde (OPA) which is the formula: ##STR2## Under mildly alkaline conditions in the presence of a thiol (R.sub.1 SH) and a primary amine (RNH.sub.2), OPA forms 1-alkylthiol-2-alkylisoindole (AAI), which is a fluorescent adduct of the formula: ##STR3##
Although the AAI fluorophores generated by the reaction of primary amines and OPA exhibit a relatively high fluorescent intensity with respect to many primary amines, it has been observed that the fluorescent intensity of the isoindole derivatives of primary amines containing an .alpha.-amido group are substantially lower. Thus, the OPA/thiol derivatizing system is of limited applicability in the assaying of femtomole quantities of peptides and proteins. Such represents a significant drawback of OPA for assaying many biological systems.
Another problem encountered with fluorogenic assaying techniques employing OPA relates to the relative instability of the 1,2-disubstituted isoindoles of certain amines such as glycine, .alpha.-aminobutyric acid and .beta.-alanine. These adducts have been observed to readily degrade into non-fluorescent products thereby placing severe time constraints on analysis.
The o-keto-aldehyde type compounds o-acetylbenzaldehyde (OAB) and o-benzoylbenzaldehyde (OBB), have also been employed as fluorogenic reagents for forming fluorescent adducts (also isoindoles) with primary amines. However, the rate of isoindole formation from OBB is too slow to make it of practical analytical value. OAB forms fluorescent isoindoles more rapidly than OBB and shows improved product stability over those formed with OPA although it is still not completely satisfactory.